Method and means for determining fitness in plants

ABSTRACT

The present invention provides methods and means for determining parent inbred plant lines with good combining ability, for determining good combinations of parent inbred plant lines capable of yielding hybrid lines with high heterosis, and further for determining the agronomical performance of different plant lines, which can be performed in vitro by determining the electron flow in the mitochondria under control and stress conditions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of application Ser. No.10/468,218 filed on Aug. 18, 2003, now U.S. Pat. No. 7,084,320 which isthe national phase of PCT International Application No. PCT/EP02/01991filed Feb. 19, 2002, which claims priority on European Application No.01200576.5 filed on Feb. 19, 2001. The entire contents of theabove-identified applications are hereby incorporated by reference.

The present invention provides methods and means for determining parentinbred plant lines with good combining ability, for determining goodcombinations of parent inbred plant lines capable of yielding hybridlines with high heterosis, and further for determining the agronomicalperformance of different plant lines, which can be performed in vitro.

BACKGROUND

A plant line can be called vigorous when this line grows vitally,healthy, is tolerant to various biotic and abiotic stresses and mostimportantly has a high yield.

The currently used method to classify plant lines according to theirgrowth and yield vigour consists in performing field trials at differentlocations. A disadvantage of field trials is that, at best, only twoexperiments can be done each year. Even when field trials are plannedvery well and deliver the appropriate data, this time constraintinterferes with the continuity of the projects and slows down theprogress.

A number of assays (mainly qualitative) have been described for use inplant tissue culture to study the effect of various stresses on thesurvival of cells or tissues (Towill and Mazur, 1975; Chen et al. 1982,Duncan and Widholm 1990, Stepan-Sarkissian and Grey, 1990; Upadhyaya andCaldwell, 1993; Enikeev et al., 1995; Ishikawa et al., 1995; Popov andVysotskaya, 1996). These are actually “viability” assays which do notmeasure the fitness or vigour of the cultures.

Chlorophyll fluorescence and fluorescence imaging may also be used tostudy the influences of stress conditions on whole plants(Lichtenthaler, 1996; Lichtentaler and Mieké, 1997). Although theseassays provide some data on the tolerance of the plant lines to certainstresses, they cannot be used to measure growth and yield vigour.

Published PCT application “WO” 97/06267 and U.S. Pat. No. 6,074,876(incorporated herein by reference) describe the use of PARP inhibitorsto improve the transformation (qualitatively or quantitatively) ofeukaryotic cells, particularly plant cells. Also described is a methodfor assessing the agronomical fitness of plants or plant material bymeasuring the electron flow in the mitochondrial electron transportchain.

None of the prior art documents describe an in vitro method allowing topredict the combining ability of parent inbred plant lines, nor do theydescribe an in vitro method allowing to determine good combinations ofparent inbred plant lines capable of yielding hybrid lines with highheterosis. Such in vitro physiological methods would represent an extratool to rapidly identify parental and hybrid lines of interest inbreeding programs and could result in a significant gain of time.

The current invention provides such methods as described in the variousembodiments and claims disclosed herein.

SUMMARY OF THE INVENTION

The invention provides a method for selecting a parent inbred plant linefrom a collection of parent lines, preferably a plant from theBrassicaceae, which upon crossing with another parent inbred plant lineis capable of yielding a hybrid plant line with high heterosis effectcomprising the steps of:

-   -   a) culturing a population of explants, preferably selected from        callus, hypocotyl explants, shoots, leaf disks and whole leaves,        particularly a hypocotyl, of each of said parent inbred plant        line of said collection under conditions which activate the        metabolism in said explant, preferably by culturing on callus        inducing medium for 1 to 10 days, particularly for about 5 days;    -   b) measuring the electron flow in the mitochondrial electron        transport chain in the population of explants, relative to the        electron flow in the mitochondrial electron transport chain in a        population of explants from a reference plant line of the same        species, preferably of the same variety, preferably by measuring        the capacity of said explant to reduce        2,3,5-triphenyltetrazolium chloride (TTC) or        3-(4,5-dimethylthiazol-2-yl)-2,3 diphenyl-2H-tetrazolium;    -   c) selecting a parent inbred line which has a high, preferably        the highest relative amount of electron flow in the        mitochondrial electron transport chain.

The method may further comprise between step a) and step b) the step ofincubating the explants for about 18 hours in a buffer comprising about25 mM K-phosphate and about 2 to 3% sucrose.

It is another object of the invention to provide a method fordetermining in vitro the agronomical fitness of a plant line comprisingthe steps of:

-   -   a) culturing a population of explants of the plant line under        conditions which activate the metabolism in said explants;    -   b) incubating, preferably for about 18 hrs, a first part of the        population of cultured explants in a buffer solution, preferably        a buffer solution comprises about 25 mM K-phosphate and about 2        to 3% sucrose;    -   c) incubating, preferably for about 18 hours, a second part of        the population of cultured explants in said buffer solution        further comprising a salicylic acid derivative capable of        generating oxidative stress in plant cells incubated in a        solution of said salicylic acid derivative, preferably        acetylsalicylic acid, particularly in a concentration of about        25 mg/L;    -   d) measuring the electron flow in the mitochondrial electron        transport chain in said first and second part of the population;        wherein the agronomically fit plants have a greater amount of        electron flow in the first part of the cultured explants than in        the second part of cultured explants.

It is yet another object of the invention to provide a method forselecting a plant line having the highest growth and yield vigour from acollection of plant lines from the same species (variety) comprising thesteps of

-   -   a) culturing a population of explants of each of the plant lines        of said collection under conditions which activate the        metabolism in said explants;    -   b) incubating, preferably for about 18 hrs, a first part of the        population of cultured explants in a buffer solution, preferably        a buffer solution comprises about 25 mM K-phosphate and about 2        to 3% sucrose;    -   c) incubating, preferably for about 18 hours, a second part of        the population of cultured explants in the buffer solution        further comprising a salicylic acid derivative capable of        generating oxidative stress in plant cells incubated in a        solution of the salicylic acid derivative, preferably        acetylsalicylic acid, particularly in a concentration of about        25 mg/L;    -   d) measuring the electron flow in the mitochondrial electron        transport chain in each of the first and second parts of the        populations; and    -   e) selecting a plant which has a high amount of electron flow in        the mitochondrial electron transport chain.

The invention also provides a method for producing a hybrid plant line,comprising the steps of:

-   -   a) assaying a collection of inbred lines of interest by        -   i) culturing a population of explants (preferably a            hypocotyl) of each of the inbred plant lines of the            collection under conditions which activate the metabolism in            the explant;        -   ii) measuring the electron flow in the mitochondrial            electron transport chain in the population of explants,            relative to the electron flow in the mitochondrial electron            transport chain in population of explants from a reference            plant line of the same species, preferably of the same            variety;    -   b) selecting a parent inbred line which has a high relative        amount of electron flow in the mitochondrial electron transport        chain when compared to other inbred lines from said collection;    -   c) crossing said selected inbred line with another inbred line;    -   d) collecting seed from said crossed selected inbred line.

Yet another object of the invention is to provide a method for producinga hybrid plant line, comprising the steps of:

-   -   a) assaying a collection of inbred lines of interest by        -   i) culturing a population of explants (preferably a            hypocotyl) of each of the parent inbred plant lines of the            collection under conditions which activate the metabolism in            the explants;        -   ii) measuring the electron flow in the mitochondrial            electron transport chain in the populations of explants,            relative to the electron flow in the mitochondrial electron            transport chain in a population of explants from a reference            plant line of the same species, preferably of the same            variety;    -   b) assaying the collection of inbred lines of interest by        -   i) culturing a population of explants of each of the parent            inbred plant lines of the collection under conditions which            activate the metabolism in the explants;        -   ii) incubating a first part of the each of the populations            of the cultured explants in a buffer solution;        -   iii) incubating a second part of each of the populations of            the cultured explants in the buffer solution, further            comprising a salicylic acid derivative capable of generating            oxidative stress in plant cells incubated in a solution of            said salicylic acid derivative, preferably acetylsalicylic            acid, particularly in a concentration of about 25 mg/L;        -   iv) measuring the electron flow in the mitochondrial            electron transport chain in each of the first and second            parts of said populations;    -   c) selecting at least one parent inbred line, preferably both        parent inbred lines, which has a high relative amount of        electron flow in the mitochondrial electron transport chain when        compared to other inbred lines from said collection and which        has a large negative difference between the measured electron        flow in the mitochondrial electron transport chain between the        second and the first part of the populations of explants;    -   d) crossing the selected inbred line with a second (inbred)        line;    -   e) collecting seed from the crossed selected inbred line.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Flow charts of the vigour assay (a) and the TTC-assay (b).

FIG. 2. Variables that influence the quality of the vigour assay. A.Influence of pre-culturing on growth medium. B. Influence of thetemperature of the preculture. C. Influence of the incubation step. D.Influence of the concentration of K-phosphate in the incubation medium.

The experiments were done with two lines of Brassica napus. The lineindicated as “control” has a seed yield comparable to the originalN90-740. The less vigorous line has a about 10% lower seed yield asscored in field trials. The metabolism of the hypocotyl explants wasactivated by culturing the explants for several days on culture mediumcontaining sucrose and hormones (A). The culture temperature has to behigh enough to activate the metabolism (B). Although the incubation stepwith phosphate buffer pH7.4 containing 2% sucrose is not obligate, itimproves the quality of the assay (C). The optimal phosphateconcentration of the incubation medium is around 25 mM (D). Each valueis the mean of three replicates with 150 explants per replicate. Theerror bars represent the standard error of the mean. In (A) (B) and (C)control line is represented by black bars, whereas the less vigorousline is represented by light bars. In (D) the values for the controlline are represented by the full line, whereas the values for the lessvigorous line are represented by the dashed line.

FIG. 3. Both the cytochromal and alternative respiratory pathwayscontribute to the reduction of TTC.

The experiments were done with two lines of Brassica napus (N90-740).The less vigorous line (light gray bars) (as scored in field trials, 90%yield versus control, and lower growth vigour) is identical to thecontrol line (dark bars) but is transgenic for the pTA29: barnase gene(Mariani et al., 1990).

SHAM alone does not seem to have a major influence on the TTC reduction.This is probably due to an overflow of the electrons from thealternative to the cytochromal respiratory pathway (Millar et al., 1995;Kumar and Kumar Acharya, 1999). The TTC-reducing capacity that remainsafter the addition of both KCN and SHAM is due to the reduction of TTCby other enzymatic activities and superoxides (Møller et al., 1988;Moore and Siedow, 1991; Raap, 1983; Seidler, 1991; Sutherland andLearmonth, 1997; Able et al., 1998). Each value is the mean of threereplicates with 150 explants per replicate. The error bars represent thestandard error of the mean.

FIG. 4. There is no correlation between the TTC-reducing capacity andthe ATP-content of five days cultured hypocotyl explants.

The hybrid lines H3 and H8 are two winter varieties of Brassica napus.H3 was scored in field trials as a good hybrid (128% yield versuscontrol line), while H8 was scored as a poor performing hybrid (91% seedyield versus control). The error bars represent the standard error ofthe mean. The dark bars represent the values measured for TTC reduction(Y1), whereas the light gray bars represent the ATP-content (Y2).

FIG. 5. The vigour assay can be used to identify lines affected in theirvigour.

The Brassica napus lines indicated as A, B, and C are derived from thespring variety Drakkar. The latter was used as a control. TheTTC-reducing capacities of the control line was set at 100%. Each valueis the mean of three replicates. The error bars represent the standarderror of the mean. When the yield of the control line is set at 100%,lines A, B and C have a yield of 86%, 84% and 95% respectively.

FIG. 6. The TTC-reducing capacity of Brassica napus parental lines andtheir hybrids. The parental lines have different genetic backgrounds(spring, winter, Chinese). The reference line is N90-740 of which theTTC-reducing capacity and yield was set to 100%. To obtain heterosis,two parental lines have to be combined of which at least one should havea high TTC-reducing capacity. Each value is the mean of three replicateswith 150 explants per replicate. The standard error was less than 7% foreach mean. Hybrid lines H1, H2, H3, H5, H8, H9 and H10 have yields ofrespectively 125%, 109%, 128%, 123%, 91%, 80% and 96%.

FIG. 7. (A) The vigour of hybrid lines can be measured by treating theexplants for a prolonged time with acetylsalicylic acid. Four hybridlines of Brassica napus (winter varieties) with different yield vigourwere tested. The TTC-reducing capacity of the four lines was similarunder control conditions. When 25 mg/l acetylsalicylic acid was added tothe incubation medium, an inverse correlation was found between theyield vigour and the TTC-reducing capacity. The line N90-740 was used asstandard for the TTC-reducing capacity and was set to 100%. Each valueis the mean of three replicates with 150 explants per replicate. Theerror bars represent the standard error of the mean. The yield of lines12, 13, 24 and 41 compared to the yield of N90-740 is respectively,110%, 104%, 114% and 118%. Dark bars represent values obtained for 0mg/l acetylsalicylic acid, light gray bars represent values obtained for25 mg/l acetylsalicylic acid.

(B) The influence of 25 mg/l acetylsalicylic acid on the TTC-reducingcapacity of 28 hybrid lines of Brassica napus (winter varieties) wasmeasured. The 28 hybrids could be classified, versus a standard controlline (set at 100%) on yield vigour in four classes: low (˜100%),moderate (104-106%), high (110-114%) and very high (>118%). The boxescontain the 25th to 75th percentile of the data. The error bars besidethe boxes represent the standard deviation with the indication of themean.

(C) Correlation between the yield and the difference in TTC reducingcapacity assayed on explants treated with acetylsalicylic acid or oncontrol explants, for different Brassica napus lines.

(D) The TTC-reducing capacity at different concentrations ofacetylsalicylic acid is different between the most and the less vigoroushybrids. The most vigorous hybrid lines show a decrease at 25 mg/lacetylsalicylic acid and again an increase at 50 mg/l acetylsalicylicacid. The lines are winter varieties with a very different geneticbackground. The TTC-reducing capacity at 0 mg/l acetylsalicylic acid isset at 100% for each line. Each value is the mean of three replicateswith 150 explants per replicate. The error bars represent the standarderror of the mean.

Dark bars represent values without acetylsalicylic acid, white bars arevalues in the presence of 25 mg/L acetylsalicylic acid, striped bars inthe presence of 50 mg/L acetylsalicylic acid, and gray bars 100 mg/Lacetylsalicylic acid.

FIG. 8. Scoring parental lines of Brassica napus for their combiningability. Parental lines can be scored for their combining ability byscoring the degree of increase or decrease of the TTC-reducing capacityin the absence and presence of acetylsalicylic acid in the incubationmedium. The lines A and B, that were identified in the breeding asexcellent combiners, have a different genetic background. Also thegenetic background from DH4, DH5, DH7 and DH10 is different from lines Aand B. DH4, DH5, DH7 and DH10 are doubled haploids derived from the sameparental line. Both the total reducing capacity at the control condition(0 mg/l acetylsalicylic acid) and the relative reducing capacities atcontrol condition (set at 100%) and after acetylsalicylic acid treatmentare plotted. The scored combining ability of DH4, DH5, DH7 and DH10 arebased on the combining ability of DH4 and DH10 with A (respectively 110%and 118% yield scores of hybrids), and the combining ability of DH5 andDH7 with B (respectively 123% and 104% yield scores of hybrids). Eachvalue is the mean of three replicates with 150 explants per replicate.The error bars represent the standard error of the mean. Combiningabilities are scored as very good (A), moderate (DH4), good (DH10), verygood (B), good (DH5) poor (DH7). Dark bars represent values obtained inthe absence of acetylsalicylic acid, set at 100% (Y1), striped barsrepresent the TTC-reducing values (%) obtained in the presence of 25mg/l acetylsalicylic acid. The light gray bars represent the absoluteTTC reducing value (Y2) scored in the absence of acetylsalicylic acid.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The invention is based on the surprising finding that heterosis inplants behaves as a dominant quantitative trait. Indeed, the inventorshave found a correlation between the amount of electron flow in themitochondrial transport chain under control conditions in the parentlines and the general fitness or vigour of the hybrid lines resultingfrom crosses between those parent lines, as determined by the differentparameters scored during field trials. These findings allow to discardcombinations of parent lines which will not yield hybrid lines with highheterosis and to proceed only with those parent lines which may yieldhybrid lines with high heterosis effect.

Furthermore, an unexpected correlation could be found between thecombining ability of parent lines and the change in the electron flow inthe mitochondrial respiration chain under stress conditions compared tothe electron flow in the mitochondrial electron transport chain undercontrol conditions (without imposing stress conditions). These findingsallow to predict the combinations of parent inbred lines which willyield hybrid lines with high heterosis effect.

Finally, an improved vigour assay based on the change in the electronflow in the mitochondrial respiration chain under stress conditionsimposed by incubation in the presence of salicylic acid or derivates,compared to the electron flow in the mitochondrial electron transportchain under controlled conditions allows a better discrimination of theresulting plant lines according to their agronomical performance.

Thus, in a first embodiment, the invention provides a method fordetermining good parental plant lines, preferably inbred lines whichupon crossing with other parent plant lines, preferably inbred plantlines is capable of yielding a hybrid plant line with high heterosis.This method comprises the steps of:

-   -   a) culturing explants of each of the parent inbred plant line of        the collection under conditions which activate the metabolism in        the explants;    -   b) measuring the electron flow in the mitochondrial electron        transport chain in the explants, relative to the electron flow        in the mitochondrial electron transport chain in the explants        from a reference plant line of the same species, preferably of        the same variety; and    -   c) selecting the parent inbred lines which have the greatest        relative amount of electron flow in the mitochondrial electron        transport chain.

As used herein, “heterosis effect” or “hybrid vigour” is used to referto the superiority of heterozygous genotypes with respect to one or morecharacters, particularly with regard to a character of interest such asyield, in comparison with the corresponding homozygotes.

Preferably, the explants are hypocotyl explants, but other explants suchas callus, shoots, leaf disks or whole leaves may be used to the sameeffect. It is further preferred that the explants should be derived fromsterile in vitro grown material that has a high respiration rate or anactive metabolism. This may be achieved by culturing or incubating theexplants first on a medium comprising a suitable carbohydrate, such assucrose, to enhance the respiration rate or metabolism. More preferably,explants are cultured on a callus inducing medium for a time sufficientto activate the metabolism, particularly for about 0 to 10 days,preferably for about 4 to 6 days, particularly for about 5 days.Preferably, the callus-inducing medium comprises sucrose, particularlyabout 2% to 3% w/w. Preferred temperature ranges for culturing theexplants are about 24 to 25° C.

Preferably, the electron flow in the mitochondrial electron transportchain is determined by measuring the capacity of the explant to reduce2,3,5 triphenyltetrazolium chloride (TTC). Although it is believed thatfor the purpose of the assays defined here, TTC is the most suitedsubstrate, other indicator molecules, such as MTT(3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl-2H-tetrazolium), can be usedto measure the electron flow in the mitochondrial electron transportchain downstream of the “ubiquinone pool”.

In another embodiment of the invention, an improved method fordetermining the vigour of a plant, i.e. for determining in vitro theagronomical fitness of a plant line is provided comprising:

-   -   a) culturing a population of explants of a parent inbred plant        line under conditions which activate the metabolism in the        explants;    -   b) incubating a first part of the cultured explants for about 18        hours in a buffer comprising about 25 mM K-phosphate and about 2        to 3% sucrose    -   c) incubating a second part of the cultured explants, preferably        for about 18 hours in a buffer comprising preferably about 25 mM        K-phosphate and about 2 to 3% sucrose and further comprising a        salicylic acid derivative capable of generating oxidative stress        in plant cells incubated in a solution of said salicylic acid        derivative, preferably in a concentration of about 25 mg/L;    -   d) measuring the electron flow in the mitochondrial electron        transport chain in said first and second part of the population.

Explants from vigourous plants have a lower electron flow in themitochondrial electron transport chain when assayed under conditionscomprising the stress with the salicylic acid derivative then whenassayed under control conditions without such imposed stress. The largerthe reduction in electron flow under stress conditions, the morevigourous the plant or plant line from which the explants were derived.When the electron flow is measured by TTC reducing capacity, and thedifference) is expressed by subtracting the data obtained from thesecond part of explants (under stress conditions) from the data obtainedfrom the first part of explants, vigourous plants should exhibit anegative score. The more negative the score, the more vigourous theplant. The Δ TTC reducing capacity may also be expressed as a percentageof the TTC reducing capacity under control conditions (mean TTC reducingcapacity under stressed conditions—mean TTC reducing capacity undercontrol conditions/mean TTC reducing capacity under control conditions).The thus obtained figure should preferably be at least about −10% butmay be as much as about −50%.

This method may be applied for selecting a plant line having the highestgrowth and yield vigour from a collection of plant lines from the samespecies, preferably from the same variety. It may also be used for theidentification of physiological conditions that affect the fitness ofthe plants (increase or decrease) or to discriminate mutant plants,cells or cell lines from wild-types.

It goes without saying that the preferred method for determining thevigour as described above may be varied within limited ranges whileretaining the improved results. Preferably the buffer is a K-phosphatebuffer in a concentration of about 25 mM, but concentrations of about 5mM to about 40 mM may be used to similar effect, as well as otherbuffers. Preferably the carbon source is sucrose in a concentration ofabout 2 to about 3% w/v but concentration ranges of about 1 to 4% may beused to the same end, as well as other carbon sources which can be usedby the plants or explants as energy sources. Preferably, the salicylicacid derivative capable of generating oxidative stress in plant cells isacetylsalicylic acid particularly in a 25 mg/L concentration range, butconcentrations as low as about 5 mg/L or as high as about 50,particularly ranging from about 20 to about 40 mg/L may also be used.

Depending on the plant species assayed, some variation in theconcentration ranges particularly those of the salicylic acid derivativemay be varied to obtain optimum results. To identify the optimumconcentration of the salicylic acid derivative, explants from a numberof reference plants or plant lines with a known yield as determined invivo, preferably in field trials, are subjected to the above describedassays using a number of concentrations of a salicylic acid derivativecapable of generating oxidative stress in a plant cell, preferablywithin the range of 5 to 50 mg/L particularly within the range of 20 to40 mg/L. The optimum concentration of salicylic acid for any given plantmay be determined as the concentration where the largest difference inTTC reducing capacity is obtained. The known yield of plant lines shouldbe correlated with the ΔTTC reducing capacity (as defined above). Theranges of buffer concentrations, temperature and incubation time may beoptimized to obtain the highest metabolic activation, as reflected bythe highest TTC reducing capacity.

To avoid any differences due to physiological conditions of the growingplants from which the explants are derived, seeds to obtain the plantsfor the assay may be primed before sowing. This may be achieved e.g. byshaking seeds for about 20 hours in sterile tap water containing 500mg/l ticarcillin disodium and selecting only those seeds that start togerminate.

The above described methods may further be used in the selection ofparent lines for the production of hybrid plants. In other words, parentlines with good combining ability may be predicted based on thefollowing method which can be performed in vitro. Thus, in anotherembodiment of the invention a method for producing a hybrid plant lineis provided, comprising the steps of:

-   -   a) assaying a collection of inbred lines of interest by        -   i) culturing a population of explants (preferably isolated            hypocotyls) of each of the parent inbred plant line of the            collection under conditions which activate the metabolism in            the explant;        -   ii) measuring the electron flow in the mitochondrial            electron transport chain in the population of explants,            relative to the electron flow in the mitochondrial electron            transport chain in population of explants from a reference            plant line of the same species, preferably of the same            variety, preferably by determining the TTC reducing            capacity;    -   b) selecting a parent inbred line which has a high relative        amount of electron flow in the mitochondrial electron transport        chain when compared to other inbred lines from the collection;    -   c) crossing the selected inbred line with another inbred line;    -   d) collecting seed from the crossed selected inbred line and        optionally, planting and growing the seed to obtain the hybrid        plants.

For more accurate prediction of the combining ability of at least oneparent line, both assay methods as described may be combined, and theparent line or lines should have a good score in both tests. Theinvention thus also provides a method for producing a hybrid plant line,comprising the steps of:

-   -   a) assaying a collection of inbred lines of interest by        -   i) culturing a population of explants (preferably            hypocotyls) of each of the parent inbred plant line of the            collection under conditions which activate the metabolism in            the explant;        -   ii) measuring the electron flow in the mitochondrial            electron transport chain in the population of explants,            relative to the electron flow in the mitochondrial electron            transport chain in population of explants from a reference            plant line of the same species, preferably of the same            variety;    -   b) assaying the collection of inbred lines of interest by        -   i) culturing a population of explants of each of the parent            inbred plant lines of the collection under conditions which            activate the metabolism in the explants;        -   ii) incubating a first part of the each of the populations            of cultured explants, preferably for about 18 hours in a            buffer preferably comprising about 20 to 30, particularly            about 25 mM K-phosphate and about 2 to 3% sucrose;        -   iii) incubating a second part of each of the populations of            the cultured explants for about 18 hours in a buffer,            preferably comprising about 20 to 30, particularly about 25            mM K-phosphate and about 2 to 3% sucrose and further            comprising a salicylic acid derivative capable of generating            oxidative stress in plant cells incubated in a solution of            the salicylic acid derivative, preferably in a concentration            of about 20 to 30 mg/L, particularly about 25 mg/L;        -   iv) measuring the electron flow in the mitochondrial            electron transport chain in each of the first and second            parts of the populations;    -   c) selecting a parent inbred line from the collection which has        a high relative amount of electron flow in the mitochondrial        electron transport chain when compared to other inbred lines        from that collection and which has a large negative difference        between the measured electron flow in the mitochondrial electron        transport chain between the second and the first part;    -   d) crossing the selected inbred line with a second inbred line;        and    -   e) collecting seed from the crossed selected inbred line and        optionally planting and growing the seed to obtain the hybrid        plants.

In a preferred embodiment, both parent lines, particularly parent inbredlines have been selected as performing good in both assays.

The methods of the invention are particularly suited for Brassicaceaeplants, particularly oilseed rape, but may be used to similar ends inother plants such as lettuce, tobacco, cotton, corn, rice, wheat,vegetable plants, carrot, cucumber, leek, pea, melon, potato, tomato,sorghum, rye, oat, sugarcane, peanut, flax, bean, sugarbeets, soya,sunflower, ornamental plants.

It has also been found that the methods described herein may be used toclassify individual plants of a particular plant variety or plant lineaccording to their agronomical fitness and yield vigour.

The following non-limiting Examples describe the particular embodimentsof the invention. Standard materials and methods for plant molecularwork are described in Plant Molecular Biology Labfax (1993) by R. D. D.Croy, jointly published by BIOS Scientific Publications Ltd (UK) andBlackwell Scientific Publications, UK. Specific experimental proceduresused in the described Examples are outlined below:

Media and Buffers

Sowing medium (medium 201): half-concentrated Murashige and Skoog salts(Murashige and Skoog, 1962), 2% sucrose, pH 5.8, 0.6% agar, 250 mg/lticarcillin disodium (Duchefa, Netherlands).

Callus inducing medium A2S3: Murashige and Skoog medium (Murashige andSkoog, 1962), 0.5 g/l Mes (pH 5.8), 3% sucrose, 40 mg/l adenine-SO4,0.5% agarose, 1 mg/l 2,4-D, 0.25 mg/l NAA, 1 mg/l BAP, 250 mg/lticarcillin disodium (Duchefa, Netherlands).

Incubation medium: 25 mM K-phosphate buffer pH5.8, 2% sucrose, 1 dropTween20 for 25 ml medium. The phosphate buffer and sucrose wereautoclaved separately. When acetylsalicylic acid was added, a stocksolution of 10 mg/ml at a pH5.8 was used.

Reaction buffer: 50 mM K-phosphate buffer pH7.4, 10 mM2,3,5-triphenyltetrazoliumchloride (=TTC) (ICN, Ohio, USA), 1 dropTween20 for 25 ml buffer. The majority of the dissolved oxygen wasremoved until a final oxygen concentration of the reaction buffer wasabout 2 mg/l. For quantifying the contribution of the cytochromal and/oralternative respiration, 1 mM KCN and/or 10 mM salicylhydroxamic acid(SHAM) were added.

Vigour Assay

Sterilization of seeds and growing of the seedlings: Seeds were soakedin 70% ethanol for 2 min, then surface-sterilized for 15 min in a sodiumhypochlorite (with about 6% active chlorine) containing 0.1% Tween20.Finally, the seeds were rinsed with 1 l of sterile distilled water.Seeds were put in 1 l vessel (Weck, Germany) containing about 75 ml ofsowing medium (10 seeds/vessel). Alternatively, the seeds were put in“in vitro vent containers” (Duchefa, The Netherlands” containing 125 mlof sowing medium (12 seeds/container). The seeds were germinated at 24°C. and 20 μEinstein/s⁻¹m⁻² with a day length of 16 h. The line N90-740was included as standard.

Priming of the seeds: If the seed quality of the tested lines wasdifferent, then the seeds were primed. This was done by shaking thesterilized seeds for about 20 hours in sterile tap water containing 500mg/l ticarcillin disodium (Duchefa, Netherlands). Only the seeds thatstarted to germinate (radicle protruded) were sown.

Preculture of the hypocotyl explants: 12 days after sowing, thehypocotyls were cut in about 7 mm segments (about 5 explants/seedling).The hypocotyl explants (25 hypocotyl explants/Optilux Petridish, FalconS1005, Denmark) were cultured for 4-5 days on medium A2S3 at 25° C. (at20 μEinstein/s⁻¹m⁻²). 150 hypocotyl explants/line/condition were used.An extra 150 hypocotyl explants were cut, these were used as blank inthe TTC-reaction (see later). Incubation step: After these 4-5 days, 150hypocotyl explants/line/condition were transferred to an OptiluxPetridish (Falcon S1005, Denmark) containing 25 ml of incubation medium,including an extra plate with the 150 hypocotyl explants that were cutat the start of the experiment (see above). This plate was used as blank(see later). The plates were incubated for about 18 hours at 24° C. inthe dark.

TTC-assay: The batches of 150 hypocotyl explants were transferred to 50ml Falcon tubes and washed with reaction buffer without TTC(2,3,5-triphenyltetrazolium chloride). 25 ml-30 ml of reaction buffer(containing 10 mM TTC) was added per tube. For the blank no TTC wasadded (for measuring the background absorption). For this the extra 150hypocotyl explants were used (see above). The explants were submerged,but no vacuum infiltration was done. The tubes were incubated upsidedown for one hour in the dark at 25° C. (no end reaction!). After thereaction, the hypocotyls were washed with deionized water. The water wasremoved and the material was frozen −70° C. for 30 min. After thawing,50 ml of technical ethanol was added per tube. The reduced TTC-H wasextracted by shaking for 1-1.5 hours. The absorption of the extract wasmeasured at 485 nm: O.D. 485 (TTC-H)=(O.D. 485+TTC)−(O.D. 485 withoutTTC). The TTC-reducing capacity of the line N90-740 was used asreference and was set at 100%.

Quantification of ATP in Hypocotyl Explants

The quantification of ATP was mainly done as described by Rawyler et al.(1999).

Material: hypocotyl explants cultured for 5 days on A2S3 medium.

ATP extraction: The hypocotyl explants were frozen with liquid nitrogen(in batches of 50 explants). Frozen hypocotyls were grinded with amortar and pistil in 6 ml of 6% perchloric acid. The extract wascentrifuged at 24,000 g (Sorvall, SS34 rotor at 14,000 rpm) for 10 min.at 4° C. The supernatant was neutralized with 5M K₂CO₃ (350 μl of 5MK₂CO₃ to 3 ml of supernatant). The KClO₄ was removed by spinning asdescribed before.

Quantitative bioluminescent determination of ATP: The ATP bioluminescentassay kit from Sigma was used (FL-AA). The luminescence was measuredwith the TD-20/20 luminometer of Turner Designs (Sunnyvale, USA).

Quantification of the Superoxide Production by the Hypocotyl Explants

The formation of superoxides by the explants was quantified by measuringthe reduction of XTT (sodium,3′-{1-[phenylamino-carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate) as described (Sutherland and Learmonth,1997; Able et al., 1998). After the incubation step of the vigour assay,the incubation medium was replaced by reaction buffer containing 1mM XTTinstead of TTC (20 ml reaction buffer for 150 hypocotyl explants). Theformation of XTT formazan was followed by measuring the absorbance ofthe medium at 470 nm.

EXAMPLE 1 Optimizing Vigour Assay Variables for a Given Plant Species

The vigour assay is generally outlined in FIG. 1. It is preferred tostart from sterile in vitro grown material that has a high respirationrate. E.g. leaf explants from greenhouse-grown plants should not be useddirectly but first be incubated with sucrose to enhance the respiration.The different components of the system, which are of importance for thereliability of the assay, were analysed using two lines derived from thespring variety N90-740 of Brassica napus. One of the lines had a seedyield comparable to the original N90-740 line and is indicated as thecontrol line. The second line had been determined by field trials ashaving a lower seed yield (about 90% of the control line) and isindicated in FIG. 2 as ‘less vigorous’ (lower seed yield, 90%, and lessvigorous growth). Except for the specific variables that were studied,the vigour assay was done as described above (see also FIG. 1B).

Culture Conditions of the Hypocotyl Explants

Freshly isolated hypocotyl explants have a low metabolic activity andrespiration rate: very low malate dehydrogenase activity and almost noTTC-reducing capacity. To activate metabolism, the explants werecultured for 0 to 10 days on callus inducing medium. The TTC-reducingcapacity was determined before incubation and after respectively 2, 4,6, and 10 days of culture. The results are shown in FIG. 2A. Theclearest relative difference between the control and less vigorous linewas measured after 4 to 6 days of culture. Often the differencesdisappear after 10 days of culture. A period of five days on callusinducing medium was chosen as the standard activation time.

Not only the duration of the culture period may influence the assay, butalso the culture conditions in general will determine how and at whichdegree the metabolism will be activated. In optimizing the vigour assayseveral variables in the culture conditions such as carbon source,carbon concentration, and temperature were evaluated. Sucrose (2-3%) wasthe best carbon source, while a culture temperature of 24-25° C. wasfound to be better than lower temperatures (FIG. 2B).

Incubation Step in K-Phosphate Buffer

Typically, the explants are incubated for about 18 hours in K-phosphatebuffer containing 2-3% sucrose. As is illustrated in FIG. 2C, thedifference in TTC-reducing capacity between the two lines with differentvigour was more pronounced when the hypocotyl explants were incubatedfor about 18 hours in K-phosphate buffer. When the TTC-assay was donedirectly on the 5 days cultured explants the difference was less clear.Moreover the incubation step allows addition of compounds such as stressinducers and enzyme inhibitors to the buffer.

In FIG. 2D the TTC-reducing capacity of the control and less vigorousline in relation to the K-phosphate concentration of the incubationmedium is depicted. Incubation in 25 mM K-phosphate resulted in thehighest values and the largest differences between the two lines.

The K-phosphate buffer and sucrose should be autoclaved separately. Forreasons that are not understood, autoclaving both together may have anadverse effect on the vigour assay.

EXAMPLE 2 Physiological Studies

Both the Cytochromal and Alternative Respiration Contribute to theReduction of TTC

Recently, it has been shown that TTC is reduced by mitochondrialdehydrogenases, particularly of Complex I. Moreover, the efficiency ofthe formazan formation depends on the activity of cytochrome oxidasewhich determines the aerobic state (Rich et al., 2001). In addition tothe cytochromal respiratory pathway, an alternative respiratory pathwayexists in plants. To analyze the relative importance of the cytochromalrespiration versus the alternative respiration in the reduction of TTC,the hypocotyl explants were put after the incubation step in reactionbuffer containing 1 mM KCN and/or 10 mM SHAM. KCN inhibits thecytochromal pathway at complex IV (cytochrome oxidase), while SHAMinhibits the alternative respiratory pathway (alternative oxidase).Kumar and Kumar Acharya (1999) described the addition of2,6-dichloro-phenol indophenol to prevent overflow of the electronsbetween the cytochromal and alternative respiratory pathways when KCNand SHAM are used as inhibitors. However, 2,6-dichloro-phenol indophenolinterfered with the TTC-assay (data not shown), for this reason2,6-dichloro-phenol indophenol was not added to the reaction buffer. Theresults of these experiments are summarized in FIG. 3. Although theobtained values do not present the correct contribution of thecytochromal and alternative respiratory pathways, they show that boththe cytochromal and alternative respiration contribute to the reductionof TTC. The TTC-reduction that remains in the presence of both KCN andSHAM, about 25%, is probably due to other pathways (Møoller et al.,1988; Moore and Siedow, 1991) and the presence of superoxides (Raap,1983; Seidler, 1991; Sutherland and Learmonth, 1997; Able et al., 1998).

The Vigour of a Plant Line is not Related to the ATP Content

The amount of TTC that is reduced in a certain time period reflects theintensity of the mitochondrial electron transport system. Therefore, itcould be that the vigour of a plant line could be measured by directlyquantifying the ATP content of the shoots or explants. Such an assaywould be convenient and quantitative.

Two lines of Brassica napus (winter varieties) with a very differentyield vigour (128% for H3, and 91% for H8) were chosen as startingmaterial. The vigour assay was performed as described but besides aTTC-assay, the ATP content of the hypocotyl explants was determined.FIG. 4 clearly shows that there is no correlation between the vigour ofthe plant line and the ATP content: in both lines the ATP content isvery similar.

EXAMPLE 3 Influence of the Quality of the Seedlot on the Vigour Assayfor Brassica Napus and Neutralization of the Seed Quality Factor by SeedPriming

Plants from the same genotype and grown at identical conditions butderived from different seed batches can vary a lot in vigour. Thisvariation in vigour is largely determined by the quality of the seedlotfrom which the plants originated (Larsen et al., 1998; Strydom and Vande Venter, 1998). This phenomenon is well known by seed companies, whotest the seedlots for their quality (read percent germination and thegrowth rate of the seedlings at optimal and suboptimal conditions)before these are introduced into the market.

To test the influence of the quality of the seedlot on the vigour assayfor Brassica napus, seedlots of two commercial lines were used asstarting material. From each line two seedlots with a high germinationefficiency (97%) but with a different seedling vigour, as judged in theseed quality tests (germination rate at 12° C. and 23° C.), were chosen.From the results of the vigour assays summarized in Table I it seemsthat the vigour assay may be influenced by the quality of the seedlotalthough to a limited extent. When the seeds were primed before sowingthe vigour scores were no longer influenced by the quality of theseedlot (Table I).

TABLE I The influence of the quality of the seedlot and the effect ofseed priming on the vigour assay of Brassica napus line A line B lot 1lot 2 lot 1 lot 2 Seed quality test (%)^(a) 95 72 97 88 Vigour 100 ± 3 92 ± 6 100 ± 2 94 ± 4 Control (%)^(b,c) Vigour 100 ± 2 101 ± 3 103 ± 399 ± 4 primed seeds (%)^(c) ^(a)as judged by scoring germination at 12°C. and 23° C. ^(b)per line, the seedlot with the highest score was setat 100% in the control condition (seeds not primed) ^(c)values representthe mean ± standard error of the mean Seedlots of a different quality(about same germination rate but giving rise to seedlings with adifferent vigour as reflected in growth rate) from two commercialvarieties (line A and line B) were used as starting material. The vigourassay was done on seedlings derived from non-treated and primed seeds.The priming was done as described in ‘Materials and Methods’.

EXAMPLE 4 The Integration of the Various Vigour Assay Methods inBrassica napus Breeding

Identification of Lines Affected in Their Vigour

In the previous Examples, two lines were used to initially set up thevigour assay. In this way optimal conditions to discriminate these twolines on the base of their TTC-reducing capacity could be defined. Theexperiment in which the lines H3 (128% yield vigour) and H8 (91% yieldvigour) were used to study the TTC-reducing capacity in relation to thetotal ATP content, already showed that the correlation between the yieldscore and the TTC-reducing capacity also holds true for these two lines.To obtain further confirmation for this correlation, three more linesthat were well-characterized in field trials, were selected. From FIG. 5it appears that the lines with a poor field performance (lines A and B)have a low TTC-reducing capacity compared to the control line and line(C). This implies that within a variety, vigour is correlated to thetotal TTC-reducing capacity: more vigorous lines having a higherTTC-reducing capacity than less vigorous lines.

The TTC-Reducing Capacity of the Parental Lines Influences the Vigour ofTheir Hybrids

From the above described experiments it appears that the vigour assaycan be used to identify weaker lines within a variety. This is ofinterest for biotechnology and breeding e.g. to test in an early phasewhether the expression of a certain transgene in a particular line couldhave an adverse effect on the overall vigour of the plant. The vigourassay would have a high added value if it would allow to identify lineswith an increased vigour, and to compare lines with a very differentgenetic background. However, each variety has its own intrinsicTTC-reducing capacity in the vigour assay (see e.g. FIG. 6). It isexpected that within a variety, lines could be compared, but the morethe varieties are genetically diverse the less the comparison of thetotal TTC-reducing capacities reflects their vigour.

To test whether the vigour assay could be used in breeding programs toevaluate lines with a different genetic background, parental lines andtheir hybrids, with a different vigour as scored in field trials (mainlyscored on seed yield), were analysed in the vigour assay. The varietyN90-740 was used as reference line and was set at 100% for TTC reducingcapacity. FIG. 6 represents an example of the results obtained from suchanalyses. The following information can be obtained from these data. Forexample, the lines P3 and P6 have the highest TTC-reducing capacity andwere scored by the breeders as the best parental lines. Well performinghybrids (with a seed yield higher than 105%) were obtained when at leastone of the parental lines had a high TTC-reducing capacity. However,from these results it cannot be concluded which one is the best hybrid(e.g. in FIG. 6, line H3 had the best field score), and cannot bepredicted which parental combinations would result in the highestheterosis.

The Best Performing Hybrid Lines can be Identified by AddingAcetylsalicylic Acid During the Incubation Period

Heterosis is a phenomenon that is not well understood. Probably, variousmolecular, biochemical, and physiological factors contribute to thehybrid vigour phenotype (Griffing, 1990; Romangnoli et al., 1990; Titoket al., 1995; Tsaftaris, 1995; Zhang et al., 1996; Milborrow, 1998;Tsaftaris and Kafka, 1998; Xiong et al., 1998; Sun et al., 1999).Tolerance to stress could be one of these factors (Cassman, 1999). Infact, stress tolerance is the plant's ability to cope with suboptimalconditions, resulting in a relatively better growth and higher yield. Ifindeed hybrids are more tolerant to less favourable growth conditions,the tolerance of lines to stress could be used as an extra criterium toclassify lines by vigour. To test this, the vigour assay was adapted byadding 0 and 25 mg/l acetylsalicylic acid to the incubation medium,imposing in this way oxidative stress on the explants (Chen et al.,1993; Dempsey and Klessig, 1994; Conrath et al., 1995; Xie and Chen,1999). An example of the results obtained in these experiments isrepresented in FIG. 7A for four hybrid lines with different yield vigour(i.e. amount of seeds produced/ha compared to a standard control line).The line with the highest seed yield (line 41) showed the strongestdecrease in TTC-reducing capacity after the acetylsalicylic acidtreatment when compared to the control condition (no addition ofacetylsalicylic acid). On the other hand the TTC-reducing capacity ofthe hybrid with the lowest seed yield (line 13) increased drasticallyafter the acetylsalicylic acid treatment. This correlation between seedyield and the degree of decrease or increase of the TTC-reducingcapacity after treatment with 25 mg/l acetylsalicylic acid versus thecontrol condition (no addition of acetylsalicylic acid), was found forall the 28 hybrid lines that were tested (FIG. 7B).

When the lines were subjected to higher concentrations ofacetylsalicylic acid (50 and 100 mg/l) it was noticed that theTTC-reducing capacity of the best performing hybrid lines increased at50 mg/l to decrease again at 100 mg/l acetylsalicylic acid. Hybrid lineswith a less pronounced heterosis didn't show at 50 mg/l acetylsalicylicacid this increase in TTC-reducing capacity (FIG. 7C). The decrease inTTC-reducing capacity by acetylsalicylic acid in those hybrids with highseed yield, could be explained by an inhibition of both the cytochromaland alternative respiratory pathways (Xie and Chen, 1999).

In summary, the decrease in TTC-reducing capacity after treatment with25 mg/l acetylsalicylic acid is more pronounced in hybrids with a highseed yield, while hybrid lines with a poor seed yield show at the sameconditions from a minor decrease to a pronounced increase. The mostheterotic hybrids, that have a significant decrease in TTC-reducingcapacity at 25 mg/l acetylsalicylic acid, show at 50 mg/lacetylsalicylic acid an increase. This increase was never observed forthe less performing hybrids.

The Increase by Acetylsalicylic Acid of the TTC-Reducing Capacity of theLess Vigorous Lines is not due to Reactive Oxygen Species but to anIncreased Electron Flow Through the Cytochromal Pathway

Although it is not described that TTC is reduced by reactive oxygenspecies, many tetrazolium dyes are reduced by superoxides (Raap, 1983;Seidler, 1991; Sutherland and Learmonth, 1997; Able et al., 1998). It iswell known that salicylic acid generates reactive oxygen species (Chenet al., 1993; Dempsey and Klessig, 1994; Conrath et al., 1995; Rao andDavis, 1999; Xie and Chen, 1999) that could possibly reduce TTC.However, in these studies the formation of superoxides was quantifiedwithin hours after the application of salicylic acid. To test whether inour system the exogenous addition of acetylsalicylic acid stillgenerates superoxides after 18 hours, the reduction of the tetrazoliumsalt XTT was measured. In the absence of the redox intermediatephenazine methosulphate (PMS), XTT is reduced only very slowly bycellular enzymes, but is reduced very efficiently by superoxides(Sutherland and Learmonth, 1997; Able et al., 1998). The results ofthese experiments revealed that after incubation for 18 hours in 25 mg/lacetylsalicylic acid, there is no significant difference in XTT-reducingcapacity between the hypocotyl explants of the four hybrid lines withdifferent vigour. In all the four lines lower amounts of XTT werereduced when the explants had been incubated for 18 hours inacetylsalicylic acid. These data indicate that after treatment for 18hours with acetylsalicylic acid, the increase in TTC-reducing capacityby the weaker lines is not due to superoxides.

The stimulating effect of salicylic acid on the alternative respiratorypathway is well known (Rhoads and McIntosh, 1992; McIntosh, 1994; VanDer Straeten et al., 1995; Lambers, 1997), although Xie and Chen (1999)describe the inhibition by salicylic acid of both the cytochromal andthe alternative oxidase pathways. In these papers the reaction of theplants to the salicylic acid applications was followed for only a fewhours. In our experiments the explants were treated for a prolongedperiod (18 hours) with acetylsalicylic acid. In the previous experimentwe showed that in the less vigorous lines superoxides are notresponsible for the enhanced TTC-reduction by acetylsalicylic acid. Forthis we checked whether an enhanced cytochromal and/or alternativerespiration could be the cause. The data obtained demonstrated that theenhanced TTC-reducing capacity after acetylsalicylic acid treatment isdue to an increased electron flow through the cytochromal and notthrough the alternative respiratory pathway and this because the byacetylsalicylic acid enhanced TTC-reduction in the weaker line (line 13)is blocked by KCN and not by SHAM.

From these experiments it can be concluded that, in the describedexperimental conditions, a prolonged treatment with acetylsalicylic acidresults: (1) in the vigorous lines in a reduction of the electron flowthrough the cytochromal pathway, (2) in the less vigorous lines in anenhancement of the electron flow through the cytochromal pathway.

The Vigour Assay can be used to Predict the Combining Ability ofParental Lines

Although it is remarkable that the vigour assay allows to identify thebest hybrids, it would be even more interesting for the breeder toidentify the best parental combinations. The experiments describedpreviously allow to predict which parental combinations will not resultin heterotic hybrids: at least one of the parents needs to have a highTTC-reducing capacity at control conditions. To discriminate between thegood and worse parental lines, the vigour assay was performed with andwithout acetylsalicylic acid treatment on parental lines with a known‘combining ability’. In FIG. 8, the relative TTC-reducing capacity undercontrol conditions and after acetylsalicylic acid treatment, as well asthe absolute TTC-reducing capacity under control conditions arerepresented. Similar to what was observed in the hybrids, the parentallines A and B with an excellent combining ability and that are used bythe breeders as general combiners, have a decreased TTC-reducingcapacity after acetylsalicylic acid treatment. The parental line DH7with a poor combining ability, shows a high increase in TTC-reducingcapacity after the acetylsalicylic acid treatment. The parental linesDH4, DH5 and DH10 with a moderate to good combining ability have no oronly a small increase in TTC-reducing capacity after acetylsalicylicacid treatment. The reason that DH4 does not combine as well as DH10with line A may be due to the lower total TTC-reducing capacity of lineDH4.

These results together with the results shown in FIG. 6 imply thatparental combinations will result in vigorous hybrids when: (1) at leastone of the parental lines and preferentially both have a highTTC-reducing capacity, and (2) the TTC-reducing capacity ofpreferentially both parental lines is reduced by the acetylsalicylicacid treatment.

REFERENCES

-   Able, A J, Guest, D I., Sutherland, M W (1998) Plant Physiol 117:    491499-   Cassman, K G (1999) Proc. Natl. Acad. Sci. USA 96: 5952-5959-   Chen, H-H, Shen, Z-Y, Li, P H (1982) Crop Science 22: 719-725-   Chen, Z, Silva, H, Kiessig, D F (1993 Science 262: 1883-1886-   Conrath, U, Chen, Z, Ricigliano, J R, Klessig, D F (1995) Proc Natl    Acad Sci USA 92: 7143-7147-   Dempsey, D A, Klessig, D F (1994) Trends Cell Biol 4: 334-338-   Duncan, D R, Widholm, J M (1990) In J W Pollard, J M Walker, eds,    Methods in Molecular Biology, Vol 6. Plant Cell and Tissue Culture.    Humana Press, Clifton, N.J., pp 29-37-   Enikeev, A G, Vysotskaya, E F, Lionova, L A, Gamburg, K Z (1995)    Russ J Plant Physiol 42(3): 372-375-   Goodwin, C J, Holt, S J, Downes, S, Marshall, N J (1995) Journal of    Immunological Methods 179: 95-103-   Graham, G I, Wolff, D W, Stuber, C W (1997) Crop Sci 37: 1601-1610-   Griffing, B (1990) Genetics 126: 753-767-   Hyslop, P A, Hinshaw, D B, Halsey Jr, W A, Schraufstätter, I U,    Sauerheber, R D, Spragg,-   R G, Jackson, J H, Cochrane, C G (1988) J Biochem Chem 263(4):    1665-1675-   Jovilet, Y, Pireaux, J-C, Dizengremel, P (1990) Plant Physiol 94:    641-646-   Kim, C S, Jung, J (1995) J Photochem Photobiol 29: 135-139-   Lin, T-Y, Markhart III, A H (1990) Plant Physiol 94: 54-58-   Kumar, S, Kumar Acharya, S (1999) Anal Biochem 268: 89-93-   Lambers, H (1997) Respiration and the alternative oxidase. In C H    Foyer, W P Quick, eds, A molecular approach to primary metabolism in    higher plants. Taylor & Francis Ltd, UK, pp 295-309-   Larsen, S U, Povisen, F V, Eriksen, E N, Pedersen, H C (1998) Seed    Sci and Technol 26: 627-641-   Lichtenthaler, H K (1996) J Plant Physiol 148: 4-14-   Lichtenthaler, H K, Miehé, J A (1997) Trends Plant Sci 2(8): 316-320-   Mcintosh, L (1994) Plant Physiol 105: 781-786-   Milborrow, B V (1998) J Exp Bot 49(324): 1063-1071-   Millar, A H, Atkin, O K, Lambers, H, Wiskich, J T, Day, D A (1995)    Physiol Plant 95: 523-532-   Minagawa, N, Koga, S, Nakano, M, Sakajo, S, Yoshimoto, A (1992) FEBS    Letters 302(3): 217-219-   Moller, I M, Bérczi, A, van der Plas, L H W, Lambers, H (1988)    Physiol Plant 72: 642-649-   Moore, A L, Siedow, J N (1991) Biochem Biophys Act 1059: 121-140-   Murashige, T, Skoog, F (1962) Physiol Plant 15: 473-497-   Musser, D A, Oseroff, A R (1994) Phytochem Photobiol 59(6): 621-626-   Popov, A S, Vysotskaya, O N (1996) Russian J Plant Physiol 43(2):    263-269-   Raap, A K (1983) Histochem J 15: 977-986-   Rao, M V, Davis, K R (1999) Plant J 17(6): 603-614-   Rawyler, A, Pavelic, D, Giainazzi, C, Oberson, J, Braendle, R (1999)    Plant Physiol 120: 293-300-   Rhoads, D M, Mcintosh, L (1992) Plant Cell 4: 1131-1139-   Ribas-Carbo, M, Aroca, R, Gonzàles-Meler, M A, Irigoyen, J J,    Sánchez-Dias, M (2000) Plant Physiol 122: 199-204-   Rich, P. R., Mischis, L. A., Purton, S., Wiskich, J. T. (2001) FEMS    Microbiol. Letters 202: 181-187-   Romangnoli, S, Maddaloni, M, Livini, C, Motto, M (1990) Theor Appl    Genet 80: 769-775-   Seidler, E (1991) Progress in Histochemistry and Cytochemistry 24:    1-86-   Stepan-Sarkissian, G, Grey, D (1990) Growth determination and medium    analysis. In J W Pollard, J M Walker, eds, Methods in Molecular    Biology, Vol 6. Plant Cell and Tissue Culture. Humana Press,    Clifton, N.J., pp 13-27-   Strydom, A, Van de Venter, H A (1998) Seed Sci and Technol 26:    579-585-   Sun, Q, Ni, Z, Liu, Z (1999) Euphytica 106: 117-123-   Sutherland, M W, Learmonth, B A (1997) Free Rad Res 27(3): 283-289-   Titok, W, Rusinova, O V, Khotyleva, L V (1995) Biol Plant 37(4):    507-513-   Towill, L E, Mazur, P (1975) Can J Bot 53: 1097-1102-   Tsaftaris, A S, Kafka, M (1998). J Crop Production 1(1): 95-111-   Tsaftaris, S A (1995) Physiol Plant 94: 362-370-   Upadhyaya, A, Caldwell, C R (1993) Environ Exp Bot 33(3): 357-365-   Van Der Straeten, D, Chaerle, L, Sharkov, G, Lambers, H, Van    Montagu, M (1995) Planta 196: 412-419-   Vanlerberghe, G C, Mcintosh, L (1996) Plant Physiol 111: 589-595-   Wagner, A M, Krab, K (1995) Physiol Plant 95: 318-325-   Xiao, J, Li, J, Yuan, L, Tanksley, S D (1995) Genetics 140: 745-754-   Xie, Z, Chen, Z (1999) Plant Physiol 120: 217-225-   Xiong, L Z, Yang, G P, Xu, C G, Zhang, Q, Saghai Maroof, M A (1998)    Mol Breeding 4: 129-136-   Zhang, Q, Zhou, Z Q, Yang, G P, Xu, C G, Liu, K D, Saghai Maroof, M    A (1996) Theor Appl Genet 93: 1218-1224

1. A method for producing a hybrid Brassica plant line, comprising thesteps of: a) assaying a collection of Brassica inbred plant lines ofinterest by i) culturing a population of explants of each of saidBrassica inbred plant lines of said collection on a medium comprisingsucrose or on callus inducing medium comprising sucrose and planthormones for a period of 0 to 10 days; ii) measuring the electron flowin the mitochondrial electron transport chain in said population ofexplants, relative to the electron flow in the mitochondrial electrontransport chain in a population of explants from a reference plant lineof the same species; b) assaying said collection of inbred lines ofinterest by i) culturing a population of explants of each of said inbredBrassica plant lines of said collection on a medium comprising sucroseor on callus inducing medium comprising sucrose and plant hormones; ii)incubating a first part of each of the populations of said culturedexplants in a buffer solution; iii) incubating a second part of each ofthe populations of said cultured explants in said buffer solution,further comprising a salicylic acid derivative capable of generatingoxidative stress in plant cells incubated in a solution of saidsalicylic acid derivative; iv) measuring the electron flow in themitochondrial electron transport chain in each of said first and secondparts of said populations; c) selecting one inbred plant line which hasa higher relative amount of electron flow in the mitochondrial electrontransport chain when compared to other inbred lines from said collectionas determined in step (a) and which has a negative difference betweenthe measured electron flow in the mitochondrial electron transport chainbetween said second and said first part as measured in step (b); d)crossing said selected inbred plant line with a second inbred plantline; e) collecting seed from said crossed selected inbred plant line.2. The method of claim 1, wherein said second inbred line has a higherrelative amount of electron flow in the mitochondrial electron transportchain when compared to other inbred lines from said collection and has anegative difference between the measured electron flow in themitochondrial electron transport chain between said second and saidfirst part.
 3. The method any one of claim 1 or 2, wherein said parts ofsaid population of cultured explants are incubated in said buffersolution for about 18 hours.
 4. The method of claim 1, wherein saidbuffer solution comprises about 25 mM K-phosphate and about 2 to 3%sucrose.
 5. The method of claim 1, wherein said salicylic acidderivative is acetylsalicylic acid.
 6. The method of claim 5, whereinsaid acetylsalicylic acid is present in said buffer solution in aconcentration of about 25 mg/L.
 7. The method of claim 1, wherein saidexplant is a hypocotyl.
 8. The method of claim 1, wherein said referenceplant line of the same species is a reference plant line of the samevariety.
 9. The method of claim 1, wherein said period is 4 to 6 days.10. The method of claim 1, wherein said period is 5 days.